The structural gene arrangement and transcript pattern in the major late transcription unit of adenovirus 2 has been determined utilizing three complementary methods of analysis: The RNA transcripts were characterized by 1) electrophoresic fractionation in agarose gels containing methyl mercury hydroxide to estimate the sizes of the functional mRNAs in cell-free translational systems; 2) RNAs were selected by annealing to viral DNA fragments immobilized on nitrocellulose to define the genomic origin of their constituent sequences; 3) hybrid-arrested translation was used to locate the protein coding sequences within the late transcriptional unit. The data obtained allowed the positioning of both the coding and noncoding regions of all the late mRNAs with respect to the viral DNA.